OBJECTIVE: To establish an accurate, simple, quick, specific and sensitive method for species identification by amplifying 12S rRNA gene with the same reaction system. METHODS: Based on the downloaded 12S rRNA gene sequences of eleven species (human, chicken, duck, goose, pig, rabbit, rat, sheep, bull, dog and goat) from GenBank, a pair of universal primers to eleven species and three pairs of specific primers to human, chicken and duck were designed. The amplicons amplified with universal primers were used for internal controls, and the amplicons amplified with specific primers were used as identification of human, chicken and duck. DNA was extracted from various samples including blood stains, fresh or freezing muscles, heat-treated muscles and hairs. Both single DNA of human, chicken or duck and mixed DNA of any two kinds of them were amplified. RESULTS: The lengths of universal amplicons were about 400 bp. The lengths of specific amplicons were 163 bp for human, 286 bp for chicken, and 374 bp for duck, respectively. No cross amplification was observed, indicating a high specificity of the specific primers. The identification rate was 100% for human, 99% for chicken, and 100% for duck, respectively. The detection sensitivity ranged from 2.5 pg to 200 pg of DNA concentration depending on species, even in mixtures of different species DNA without interference. CONCLUSION The method established could identify different species under the same reaction system.
Animals ; Blood ; Cattle ; DNA/analysis* ; Dogs ; Forensic Genetics ; Hot Temperature ; Humans ; Polymerase Chain Reaction/veterinary* ; Poultry/genetics* ; RNA, Ribosomal/genetics* ; Rabbits ; Rats ; Sheep ; Species Specificity ; Swine

OBJECTIVETo investigate the patency efficacy of small diameter artificial vascular graft with eNOS gene transfected endothelial cells in canine.

METHODSBy using the two steps high pressure injection, eNOS gene transfected endothelial cells were planted on artificial vascular graft with a diameter of 3 mm. The artificial vascular grafts were transplanted into canine femoral artery, and the patency of the artery was observed through digital subtracted angiography (DSA) and electron telescope 1, 4, 12 and 24 weeks after the operation.

RESULTSThe adherence rate of eNOS gene transfected endothelial cells to artificial vascular grafts was up to 90%. For 10 days, the cells extended and formed a continuous intima. One week after the operation, 3/9 grafts were obstructed in the group of simple artificial vascular grafts; 4 weeks after, all of grafts (9/9) were obstructed in the group of simple artificial vascular grafts and almost half grafts (5/10, 4/9) in un-transfected groups were obstructed; 12 weeks after, all of the grafts were obstructed in the group of simple artificial vascular grafts and in un-transfected groups (9/9, 9/10); 24 weeks after, all of the grafts were obstructed in the groups of un-transfected artificial vascular grafts, while nearly all of grafts (8/10) were open in eNOS groups. Electron microscope scanning showed that endothelial cells of artificial vascular grafts in eNOS transfected groups arranged closely and formed a continuous intima; only few red blood cells, leucocytes, platelets deposited at the surface of endothelial cells in the artificial vascular grafts.

CONCLUSIONSThe patency efficacy of eNOS gene transfected endothelial cells planting on artificial vascular graft is satisfactory. It could provide an experimental basis for further clinical application of the artificial vascular grafts.

Animals ; Blood Vessel Prosthesis ; Blood Vessel Prosthesis Implantation ; Cells, Cultured ; Dogs ; Endothelial Cells ; cytology ; metabolism ; Female ; Genetic Vectors ; Male ; Nitric Oxide Synthase ; genetics ; Random Allocation ; Transfection

Toxoplasmosis, caused by Toxoplasma gondii, is a parasitic zoonosis with worldwide distribution. The present study investigated the prevalence of T. gondii in dogs in Zhanjiang city, southern China, using both serological and molecular detection. A total of 364 serum samples and 432 liver tissue samples were collected from the slaughter house between December 2012 and January 2013 and were examined for T. gondii IgG antibody by ELISA and T. gondii DNA by semi-nested PCR based on B1 gene, respectively. The overall seroprevalence of T. gondii IgG antibody was 51.9%, and T. gondii DNA was detected in 37 of 432 (8.6%) liver tissue samples. These positive DNA samples were analyzed by PCR-RFLP at 3'- and 5'-SAG2. Only 8 samples gave the PCR-RFLP data, and they were all classified as type I, which may suggest that the T. gondii isolates from dogs in Zhanjiang city may represent type I or type I variant. This study revealed the high prevalence of T. gondii infection in dogs in Zhanjiang city, southern China. Integrated measures should be taken to prevent and control toxoplasmosis in dogs in this area for public health concern.
Animals ; Antibodies, Protozoan/blood ; China/epidemiology ; Dog Diseases/epidemiology/*parasitology ; Dogs ; Female ; Genotype ; Liver/parasitology ; Male ; Toxoplasma/classification/genetics/immunology/*isolation & purification ; Toxoplasmosis, Animal/blood/epidemiology/*parasitology

Animals ; Arterial Occlusive Diseases ; physiopathology ; therapy ; Dogs ; Genetic Therapy ; Hepatocyte Growth Factor ; genetics ; Hindlimb ; blood supply ; Ischemia ; physiopathology ; therapy ; Male ; Plasmids

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Concanavalin A/pharmacology ; DNA, Complementary/*chemistry ; Dogs/blood/genetics/*immunology ; Interleukin-3/chemistry/*genetics ; Interleukin-6/chemistry/*genetics ; Leukocytes, Mononuclear/chemistry/drug effects ; Molecular Sequence Data ; Open Reading Frames/genetics ; Protein Sorting Signals/genetics ; RNA/blood/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid

OBJECTIVETo investigate the effect of shenfu injection on canine with cardiogenic shock and the possible mechanism.

METHODCardiogenic shock model of canine was established by ligating left anterior descending (LAD) of coronary artery. The 15 canines with cardiogenic shock were randomly divided in to glucose injection group, shenfu injection group and sham-operated group. The hemodynamics parameters were monitored. Plasma TNF-alpha and IL-1beta levels were measured by radioimmunoassay. Expression of TNF-alpha mRNA and IL-1beta mRNA in myocardium were detected by RT-PCR.

RESULTFollowing cardiogenic shock, the mean artery pressure (MAP), left ventricular systolic pressure (LVSP), ventricular pressure rise ratio during systolic period (+ dp/dt(max)), and ventricular pressure decay ratio during diastolic period (- dp/dt(max)) decreased significantly; the plasma TNF-alpha and IL-1beta levels and the expression of TNF-a mRNA and IL-1beta mRNA in myocardium increased significantly. In shenfu injection group, MAP, LVSP and +/- dp/dt(max) increased significantly and plasma TNF-alpha and IL-1beta levels decreased significantly. In glucose injection group, MAP, LVSP, +/- dp/dt(max) and plasma TNF-alpha and IL-1beta levels had not changed significantly. The expression of TNF-alpha mRNA and IL-1beta mRNA in myocardium were significantly lower in shenfu injection group than those in glucose injection group.

CONCLUSIONShenfu injection probably can decrease over-exprssion of TNF-alpha mRNA and IL-1beta mRNA on transcription platform. Shenfu injection counteract cardiogenic shock, relieve myocardium damage and improve hemodynamics through inhibiting overproduction of TNF-alpha and IL-1beta.

Aconitum ; chemistry ; Animals ; Cytokines ; biosynthesis ; blood ; genetics ; Dogs ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Female ; Hemodynamics ; drug effects ; physiology ; Injections, Intravenous ; Interleukin-1beta ; biosynthesis ; blood ; genetics ; Male ; Myocardium ; metabolism ; Panax ; chemistry ; Phytotherapy ; Plants, Medicinal ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Shock, Cardiogenic ; physiopathology ; prevention & control ; Tumor Necrosis Factor-alpha ; biosynthesis ; blood ; genetics

OBJECTIVE: To identify the species of biological samples by amplifying intron 8 of the TP53 gene. METHODS: Collected the bloodstains or muscle tissues of 14 kinds of common animals and human. DNA were quantified after extraction. The PCR amplification products were analyzed by PAGE and silver staining. RESULTS: The human and monkey have one amplification band of 460 bp while the eel, fish, frog, duck, rabbit, cat, mouse, cavy, pig, ox and sheep could amplify different unspecial bands. No amplification products were found in chicken and dog. CONCLUSION The method of species identification by amplifying intron 8 of the TP53 gene is simple and sensitive.
Animals ; Blood Stains ; Cats ; Cattle ; Chickens ; DNA/genetics* ; DNA Primers ; Dogs ; Electrophoresis, Polyacrylamide Gel ; Fishes ; Forensic Medicine ; Gene Amplification ; Genes, p53 ; Humans ; Introns/genetics* ; Mice ; Polymerase Chain Reaction/methods* ; Rabbits ; Species Specificity ; Staining and Labeling

AIMTo investigate the effect of androgen on the proliferation, differentiation and regression of canine prostatic stromal cells in vivo and human stromal cells in vitro.

METHODSTwenty-two dogs, including 15 normal prostate dogs and 7 prostatic hyperplasia dogs, had their serum concentration of testosterone and estrodiol determined by radioimmunoassay before and after castration. The expression of androgen receptor (AR) and estrogen receptor (ER) in the prostate were analysed by immunohistochemistry and semi-quantitative RT-PCR before and after castration. Light microscopy, transmission electron microscopy and TUNEL assay were carried out successively before and after castration to evaluate the prostatic histomorphology. In vitro serum-free cell cultures from human prostatic stroma were established and exposed to dihydrotestosterone (DHT). The proliferation of the cell culture was detected by MTT assay. The expression of TGFbgr, bFGF, AR, and smooth muscle cell (SMC) specific proteins (myosin and/or smoothelin) were detected using immunohistochemistry and RT-PCR. The differentiation from fibroblasts to smooth muscle cells was deduced by measuring the expression of SMC specific proteins.

RESULTSBefore castration, the serum concentrations of testosterone and estrodiol were not statistically different between normal and hyperplasia groups. Following castration, the serum concentration of testosterone decreased rapidly in 2 days, and the concentration of estrodiol had no significant change compared with the pre-castration data. In the prostate, AR was presented in both the epithelial and stromal cells and the AR mRNA level was higher in hyperplasia than in normal prostate tissues (P<0.05). While ER predominantly existed in the prostate stromal cells and the ER mRNA had no difference between the hyperplasia and the normal group. Within the early phase of castration (

CONCLUSIONThe whole prostate gland is an androgen-sensitive organ with both the epithelium and stroma under the control of androgen. Androgen may direct the proliferation, differentiation and regression of stromal cells by regulating the expression of TGFbgr, bFGF, AR and smooth muscle cell specific proteins.

Animals ; Biomarkers ; Cell Differentiation ; drug effects ; physiology ; Cell Division ; drug effects ; physiology ; Cells, Cultured ; Dihydrotestosterone ; pharmacology ; Dogs ; Estradiol ; blood ; Fibroblast Growth Factor 2 ; genetics ; pharmacology ; Gene Expression ; Humans ; Male ; Muscle, Smooth ; cytology ; physiology ; Orchiectomy ; Prostate ; cytology ; physiology ; Prostatic Hyperplasia ; physiopathology ; RNA, Messenger ; analysis ; Receptors, Androgen ; genetics ; Receptors, Estrogen ; genetics ; Stromal Cells ; cytology ; physiology ; Testosterone ; blood ; Transforming Growth Factor beta ; genetics ; pharmacology

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.
Animals ; Blood/*parasitology ; Brugia/classification/genetics/*isolation & purification ; Cats ; Culicidae/*parasitology ; Dirofilaria immitis/classification/genetics/*isolation & purification ; Dogs ; Humans ; Male ; Parasitology/*methods ; RNA, Helminth/genetics ; RNA, Ribosomal, 5S/genetics ; Real-Time Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Transition Temperature ; Wuchereria bancrofti/classification/genetics/*isolation & purification

OBJECTIVETo investigate the molecular genetic basis of the B3 variant of ABO blood group system with mixed-field hemagglutination in Chinese.

METHODSSerological techniques were performed to characterize the erythrocyte phenotype of two discrepant samples. A sequential agglutination method and 13 short tandem repeat (STR) loci were tested to exclude the possibility of exogenous or endogenous DNA chimera. Mutations in exons 6 and 7, including partial intron of the ABO gene, were screened by polymerase chain reaction and DNA sequencing. Haplotypes of the two individuals were also analyzed by sequencing.

RESULTSA mixed-field hemagglutination of RBCs with anti-B and anti-AB antibodies was detected in the two unrelated individuals. Exogenous ABO-incompatible RBC transfusion and endogenous genetic chimera were excluded by sequential agglutination method and STR. The ABO phenotypes of the two individuals were classified as A1B3 according to the ABO subgroup definition. The sequence region from intron 5 to 3'-UTR of the B allele was identical to that of ABO*B101 allele, except for a T to C substitution at nucleotide position 425 in exon 7. This substitution resulted in an amino acid change of M142T in the B glycosyltransferase.

CONCLUSIONA novel B allele with 425T>C substitution resulting in B3 subgroup was identified in two Chinese individuals.

1,4-alpha-Glucan Branching Enzyme ; genetics ; ABO Blood-Group System ; genetics ; Alleles ; Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Asian Continental Ancestry Group ; genetics ; Cattle ; DNA Mutational Analysis ; Dogs ; Humans ; Methionine ; genetics ; Mice ; Molecular Sequence Data ; Mutation ; Phenotype ; Rats ; Sequence Alignment ; Sequence Analysis, DNA ; Threonine ; genetics